Connexins are integral membrane proteins that form intercellular channels called gap junctions. Our long-term goal is to understand the mechanisms responsible for chemical regulation of gap junctions. Here, we focus on the molecular steps involved in the closure of Cx43 by low pHi, or by exposure to insulin. We have previously proposed a particle-receptor model for the chemical regulation of Connexin43. In this project, we will focus on the following specific aims: 1) To establish the prevalence of the particle-receptor model of chemical gating among the connexin family. We propose that the particle-receptor mechanism can be demonstrated in a sub-group of connexin proteins. Recognizing such a subgroup will help in the identification of key structural features, and the possible application of peptide-directed strategies to interfere with the chemical gating of selected gap junctions. 2) To determine the level of promiscuity of the particle-receptor interaction. We hypothesize that the structure that serves as a receptor for the particle is preserved among connexins. Therefore, the CT domain from one connexin may interact with a gap junction channel formed by another connexin. If present, these hetero-domain interactions would be a higher mechanism of regulation in heteromeric gap junctions. 3) To identify structural and functional features of the receptor for the Cx43 chemical gate. We propose that regions of the CL (including histidine 95) are part of the chemical gate receptor. We will also determine whether the receptor involves polar amino acids that are subject to the electrical transjunctional field created during a voltage pulse. 4) To study the possible interaction between pH gating and insulin- induced uncoupling of Cx43. We have found that these two forms of chemical gating share a common molecular path. Here we will determine whether in combination the two mechanisms have an additive, synergistic, or antagonistic effect. These are the first steps toward understanding the multiple interactions between regulators of Cx43 that may be active at once in a multicellular system during pathological conditions such as ischemia.